• Journal of Oral Medicine and Pain
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• v.35 no.3
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• pp.165-175
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• 2010

Valproic acid (VPA) is a well-known anticonvulsive agent and has been used in the treatment of epilepsy for almost 30 years. VPA emerged in 1997 as an antineoplastic agent as well, when findings indicated the substance inhibited proliferation and induced differentiation of primitive neuroectocdermal tumor cells in vivo (Cinatl et al., 1997). Antitmor activity of VPA is associated with its targeting histone deacetylases. Bile acids and their synthetic derivatives induced apoptosis in various kinds of cancer cells and anticancer effects. It has been reported that the synthetic chenodeoxycholic acid (CDCA) derivatives showed apoptosis-inducing activity on various cancer cells in vitro. This study was undertaken to investigate the synergistic apoptotic effect of co-treatment with the histone deacetylases inhibitor, VPA and a CDCA derivative, HS-1200 on human osteosarcoma (HOS) cells. Cell viability was evaluated by trypan-blue exclusion. Induction and augmentation of apoptosis were confirmed by Hoechst staining, flow cytometry (DNA hypoploidy and MMP change), Westen blot analysis and immunofluorescent staining. In this study, HOS cells co-treated with VPA and HS-1200 showed several lines of apoptotic manifestation such as nuclear condensations, the reduction of MMP, the decrease of DNA content, the release of cytochrome c into cytosol, the translocation of AIF onto nuclei, and activation of caspase-7, caspase-3 and PARP whereas each single treated HOS cells did not. Although the single treatment of 1 mM VPA or $25\;{\mu}M$ HS-1200 for 48 h did not induce apoptosis, the co-treatment of them induced prominently apoptosis. Therefore our data provide the possibility that combination therapy of VPA and HS-1200 could be considered as a novel therapeutic strategy for human osteosarcoma.

• Journal of Life Science
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• v.25 no.11
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• pp.1214-1222
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• 2015

The purpose of this study was to determine the effect of Pueraria lobate-root based combination supplementation containing Rehmannia glutinosa and exercise on histone modification in ovariectomized rat hindlimb skeletal muscle. Sixty rats were fed with high fat diet and randomly assigned into the following groups for 8 weeks: 1)HSV; High fat+Sedentary+Vehicle, 2)HSP; High fat+Sedentary+PR, 3)HSH; High fat+Sedentary+Estradiol, 4)HEV; High fat+Ex+Vehicle, 5)HEP; High fat+Ex+PR, 6)HEH; High fat+Ex+Estradiol. Exercise consisted of low intensity treadmill exercise(1-4th wk:15 m/min for 30 min, 5-8th wk: 18 m/min for 40 min, 5 times/week). The result of this study showed that exercise and Pueraria and Rehmannia glutinosa intake suppressed weight gain. Furthermore, exercise and Pueraria and Rehmannia glutinosa intake increased muscle mass. This study observed H3K9 acetylation and demethylation in plantaris muscle in exercised group, but no difference in soleus muscle. To test whether the decrease in HDAC4, HDAC5 and G9a mRNA levels after exercise and Pueraria/Rehmannia glutinosa intake, HDAC4, HDAC5 and G9a mRNA levels were determined by real-time PCR. Only exercise induced HDAC5 and G9a mRNA reduction in plantaris muscle, but not in soleus muscle. In conclusion, these data demonstrates that exercise and Pueraria/Rehmannia glutinosa intake effect on body compositions. These changes are regulated by epigenetic modifications, such as histone acetylation and methylation. Future studies should focus on gene-specific epigenetics and other epigenetic mechanism for Pueraria/Rehmannia glutinosa intake.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.18
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• pp.7849-7855
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• 2014

Purpose: To investigate the effect of deacetylase inhibitory trichostatin A (TSA) on anti HepG2 liver carcinoma cells and explore the underlying mechanisms. Materials and Methods: HepG2 cells exposed to different concentrations of TSA for 24, 48, or 72h were examined for cell growth inhibition using CCK8, changes in cell cycle distribution with flow cytometry, cell apoptosis with annexin V-FTIC/PI double staining, and cell morphology changes under an inverted microscope. Expression of ${\beta}$-catenin, HDAC1, HDAC3, H3K9, CyclinD1 and Bax proteins was tested by Western blotting. Gene expression for ${\beta}$-catenin, HDAC1and HDAC3 was tested by q-PCR. ${\beta}$-catenin and H3K9 proteins were also tested by immunofluorescence. Activity of Renilla luciferase (pTCF/LEF-luc) was assessed using the Luciferase Reporter Assay system reagent. The activity of total HDACs was detected with a HDACs colorimetric kit. Results: Exposure to TSA caused significant dose-and time-dependent inhibition of HepG2 cell proliferation (p<0.05) and resulted in increased cell percentages in G0/G1 and G2/M phases and decrease in the S phase. The apoptotic index in the control group was $6.22{\pm}0.25%$, which increased to $7.17{\pm}0.20%$ and $18.1{\pm}0.42%$ in the treatment group. Exposure to 250 and 500nmol/L TSA also caused cell morphology changes with numerous floating cells. Expression of ${\beta}$-catenin, H3K9and Bax proteins was significantly increased, expression levels of CyclinD1, HDAC1, HDAC3 were decreased. Expression of ${\beta}$-catenin at the genetic level was significantly increased, with no significant difference in HDAC1and HDAC3 genes. In the cytoplasm, expression of ${\beta}$-catenin fluorescence protein was not obvious changed and in the nucleus, small amounts of green fluorescence were observed. H3K9 fluorescence protein were increased. Expression levels of the transcription factor TCF werealso increased in HepG2 cells following induction by TSA, whikle the activity of total HDACs was decreased. Conclusions: TSA inhibits HDAC activity, promotes histone acetylation, and activates Wnt/${\beta}$-catenin signaling to inhibit proliferation of HepG2 cell, arrest cell cycling and induce apoptosis.

• Journal of Oral Medicine and Pain
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• v.36 no.1
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• pp.11-20
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• 2011

Valproic acid (VPA) is a well-known anticonvulsive agent and has been used in the treatment of epilepsy for almost 30 years. VPA emerged in 1997 as an antineoplastic agent. And it is known that antitmor activity of VPA is associated with its targeted at histone deacetylases. 17AAG, Inhibition of HSP90 leads to the proteasome degradation of the HSP90 client proteins, such as Akt, Raf/Ras, Erk, VEGF, cyclin D and p53, and causes potent antitumor activity. It is reported that 17AAG-induced HSP90 inhibition results in prevention of cell proliferation and induction of apoptosis in several types of cancer. This study was undertaken to investigate the synergistic apoptotic effect of co-treatment with the histone deacetylases inhibitor, VPA and the HSP90 inhibitor, 17AAG on human osteosarcoma (HOS) cells. Cell viability was evaluated by trypan-blue exclusion. Induction and augmentation of apoptosis were confirmed by Hoechst staining, flow cytometry (DNA hypoploidy and MMP change), Westen blot analysis and immunofluorescent staining. In this study, HOS cells co-treated with VPA and 17AAG showed several lines of apoptotic manifestation such as nuclear condensations, the reduction of MMP, the decrease of DNA content, the release of cytochrome c into cytosol, the translocation of AIF onto nuclei, and activation of caspase-3, caspase-7 and PARP whereas each single treated HOS cells did not. Although the single treatment of 1 mM VPA or 0.5 ${\mu}M$ 17AAG for 48 h did not induce apoptosis, the co-treatment with them induced prominently apoptosis. Therefore our data in this study provide the possibility that combination therapy with VPA and 17AAG could be considered as a novel therapeutic strategy for human osteosarcoma.

• Radiation Oncology Journal
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• v.28 no.4
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• pp.219-223
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• 2010

Purpose: To test the radiosensitizing effect of the newly synthesized novel histone deacetylase inhibitor, SK-7041 in vivo. Materials and Method: The RIF-l cell line was implanted into the back of a 6-week-old female C3H mouse, intradermally, The mice were grouped into control, drug, radiation (RT), and RT+drug group. SK-7041, 4 mg/kg was administered intraperitoneally for six cycles every 12 hours for mice in the drug and RT+drug group, An identical volume of phosphate buffered saline (PBS) was administered at the same frequency to mice in the control and RT groups. A single 5 Gy fraction was delivered to mice in RT and RT+drug group 6 hours after the fourth delivery. The volume of the implanted tumor was measured every 2~3 days to formulate the growth delay curve. Results: For the control, drug, RT, and RT +drug groups, the average duration for implanted tumor to reach a volume of $1,500mm^3$ was 10 days, 10 days, 9 days, and 12 days, respectively. Moreover, the tumor volume on D14 was $276.7mm^3$, $279.9mm^3$, $292.5mm^3$, and $185.5mm^3$, respectively (p=0.0004). The difference for the change in slope for the control and drug versus the RT and RT+drug groups were borderline significant (p=0.0650). Conclusion: The results of this study indicate that SK-7041 has a radiosensitizing effect for the RIF-1 cell line in vivo at a low concentration and this effect may be synergistic. Implementing this result to clinical trial is warranted.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.64 no.4
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• pp.384-394
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• 2019

We have investigated the effects of ambient temperature on the growth of wheat in Korea. The differences in the growth phase of wheat were compared according to the temperature treatment. The productive tiller number and dry weight were decreased in a plot under a higher temperature treatment. We found that the growth of Jinpum was different from that of the alternative wheat cultivars, which were bred in Korea, at 50 days after treatment. While the Jinpum wheat grown at 17℃ showed vegetative stage growth, that grown in the 23℃ growth chamber entered the heading and flowering stage. The differences in the expression of 16 genes known to be involved in high-temperature responses were checked by using Jinpum wheat 50 days after two temperature treatments (17℃ and 23℃), which showed apparent differences in expression between the higher and lower temperatures during the growth phase. In the 23℃ treatment samples, the genes with increased expression were HSP70, HSP101, VRN2, ERF1, TAA1, YUCCA2, GolS, MYB73, and Histone H2A, while the genes with decreased expression were VRN-A1, DREB2A, HsfA3, PIF4, PhyB, HSP17.6CII, rbcL, and MYB73. YUCCA2, HSP101, ERF1, and VRN-A1 showed a significant difference in gene expression between lower- and higher-temperature conditions. Overall, combining the means of the expression of various genes involved in thermosensing, vernalization, and abiotic stresses, it is possible to conclude that different sets of genes are involved in vernalization and summer depression of wheat under long term, high ambient temperature conditions.

• Radiation Oncology Journal
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• v.23 no.2
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• pp.116-122
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• 2005

Purpose: Histone deacetylase inhibitors (HDIs) are emerging as potentially useful components of anticancer therapy and their radiosensitizing effects have become evident. Specific HDAS are now available that preferentially inhibit specific HDAC classes; TSA inhibits Class I and II HDACs, and SK7041 inhibits Class I HDACs. Materials and methods: We tested the differential radiosensitization induced by two different classes of HDIs in HeLa cells. We next tested the hypothesis that p53 expression in cancer cells may influence the susceptibility to HDIs by using pharmacologic modification of the p53 status under an isogenic background. Results: It is interesting that p53 expression in the HeLa cells clearly increased the degree of radio-sensitization by TSA compared to that of the class I specific inhibitor SK7041. This suggests that p53 may, in part, be responsible for the mechanistic role for the greater radiosensitization induced by Class I & II inhibitors compared to that of the class I specific inhibitors. Thus, these studies are useful in distinguishing between events mediated solely by the Class I HDACS versus those events involving the other classes of HDACS as well. Conclusion: The anticancer efficacy of targeting Class I and II HDACS, in conjunction with radiation therapy, may be further enhanced by the restoration of p53 expression.

• Journal of Life Science
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• v.30 no.12
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• pp.1092-1100
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• 2020

The six-transmembrane epithelial antigen of prostate 4 (Steap4) is a metalloreductase that plays a role in intracellular iron and cupper homeostasis, inflammatory response, and glucose and lipid metabolism. Previously, Steap4 has been reported to stimulate adipocyte differentiation; however, the underlying mechanisms of this action remain unexplored. In the present study, we investigated the molecular mechanisms involved in Steap4-induced adipocyte differentiation using 3T3-L1 cells, immortalized brown adipocyte (iBA) cells, and mouse embryonic fibroblast C3H10T1/2 cells. The knockdown of Steap4 using adenovirus-containing shRNA attenuated mitotic clonal expansion (MCE), as evidenced by the impaired proliferation of 3T3-L1 cells, iBA cells, and C3H10T1/2 cells within 48 hr after adding the differentiation medium. Steap4 knockdown downregulated G1/S phase transition-related cell cycle regulators (including cyclin A and cyclin D) and upregulated cell cycle inhibitors (including p21 and p27). Furthermore, Steap4 knockdown inhibited the phosphorylation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and Akt. Moreover, Steap4 knockdown repressed the expression of early adipogenic activators, such as CCAAT-enhancer-binding protein β (C/EBPβ) and Kruppel-like factor family factor 4 (KLF4). On the other hand, Steap4 knockdown stimulated the expression of adipogenic inhibitors, including KLF2, KLF3, and GATA2. The overexpression of Steap4 using an adenovirus removed the repressive histone marks H3K9me2 and H3K9me3 on the promoter of C/EBPβ. These results indicate that Stepa4 stimulates adipocyte differentiation through the induction of MCE and the modulation of early adipogenic transcription factors, including C/EBPβ, during the early phase of adipocyte differentiation.

• Tuberculosis and Respiratory Diseases
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• v.56 no.5
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• pp.465-484
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• 2004

Background : Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) inhibits the proliferation of non-small cell lung cancer (NSCLC) cells by inducing apoptosis. Methods : In this study, we investigated whether hypermethylation of IGFBP-3 promoter play an important role in the loss of IGFBP-3 expression in NSCLC. We also studied the mechanisms that mediate the silencing of IGFBP-3 expression in the cell lines which have hypermethylated IGFBP-3 promoter. Results : The IGFBP-3 promoter has hypermethylation in 7 of 15 (46.7%) NSCLC cell lines and 16 (69.7%) of 23, 7 (77.8%) of 9, 4 (80%) of 5, 4 (66.7 %) of 6, and 6 (100%) of 6 tumor specimens from patients with stage I, II, IIIA, IIIB, and IV NSCLC, respectively. The methylation status correlated with the level of protein and mRNA in NSCLC cell lines. Expression of IGFBP-3 was restored by the demethylating agent 5'-aza-2'-deoxycytidine (5'-aza-dC) in a subset of NSCLC cell lines. The Sp-1/ Sp-3 binding element in the IGFBP-3 promoter, important for promoter activity, was methylated in the NSCLC cell lines which have reduced IGFBP-3 expression and the methylation of this element suppressed the binding of the Sp-1 transcription factor. A ChIP assay showed that the methylation status of the IGFBP-3 promoter influenced the binding of Sp-1, methyl-CpG binding protein-2 (MeCP2), and histone deacetylase (HDAC) to Sp-1/Sp-3 binding element, which were reversed by by 5'-aza-dC. In vitro methylation of the IGFBP-3 promoter containing the Sp-1/Sp-3 binding element significantly reduced promoter activity, which was further suppressed by the overexpression of MeCP2. This reduction in activity was rescued by 5'-aza-dC. Conclusion : These findings indicate that hypermethylation of the IGFBP-3 promoter is one mechanism by which IGFBP-3 expression is silenced and MeCP2, with recruitment of HDAC, may play a role in silencing of IGFBP-3 expression. The frequency of this abnormality is also associated with advanced stages among the patients with NSCLC, suggesting that IGFBP-3 plays an important role in lung carcinogenesis/progression and that the promoter methylation status of IGFBP-3 may be a marker for early molecular detection and/or for monitoring chemoprevention efforts.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.2
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• pp.266-277
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• 2014

Somatic cell nuclear transfer (SCNT) has generally demonstrated that a differentiated cell can convert into a undifferentiated or pluripotent state. In the SCNT experiment, nuclear reprogramming is induced by exposure of introduced donor nuclei to the recipient cytoplasm of matured oocytes. However, because the efficiency of SCNT still remains low, a combination of SCNT technique with the ex-ovo method may improve the normal development of SCNT embryos. Here we hypothesized that treatment of somatic cells with extracts prepared from the germinal vesicle (GV) stage Siberian sturgeon oocytes prior to their use as nuclear donor for SCNT would improve in vitro development. A reversible permeability protocol with $4{\mu}g/mL$ of digitonin for 2 min at $4^{\circ}C$ in order to deliver Siberian sturgeon oocyte extract (SOE) to porcine fetal fibroblasts (PFFs) was carried out. As results, the intensity of H3K9ac staining in PFFs following treatment of SOE for 7 h at $18^{\circ}C$ was significantly increased but the intensity of H3K9me3 staining in PFFs was significantly decreased as compared with the control (p<0.05). Additionally, the level of histone acetylation in SCNT embryos at the zygote stage was significantly increased when reconstructed using SOE-treated cells (p<0.05), similar to that of IVF embryos at the zygote stage. The number of apoptotic cells was significantly decreased and pluripotency markers (Nanog, Oct4 and Sox2) were highly expressed in the blastocyst stage of SCNT embryos reconstructed using SOE-treated cells as nuclear donor (p<0.05). And there was observed a better development to the blastocyst stage in the SOE-treated group (p<0.05). Our results suggested that pre-treatment of cells with SOE could improve epigenetic reprogramming and the quality of porcine SCNT embryos.